| Human Molecular Genetics | Pages |
©1999 Oxford University Press |
Properties of connexin26 gap junctional proteins derived from mutations associated with non-syndromal heriditary deafness
Introduction
Results
Functionality of cells expressing mutated connexins
Expression of the mutated connexins in COS-7 cells
Cell-free synthesis of Cx26 mutations
Oligomeric status of connexin26 mutations
Discussion
Materials And Methods
Construction of Cx26 mutations
Functional analysis of cells expressing mutated Cx26
Analysis of the oligomeric state of mutated connexons
Acknowledgements
References
Properties of connexin26 gap junctional proteins derived from mutations associated with non-syndromal heriditary deafness
Received June 18, 1999; Revised and Accepted September 13, 1999
Three point mutations of the connexin26 (GJB2) gene associated with hereditary deafness were studied using in vitro expression systems. Mutation M34T results in an amino acid substitution in the first transmembrane domain of the connexin protein, W77R is located in the second transmembrane domain and W44C is in the first extracellular loop. Wild-type and mutated connexin vectors were constructed and transfected into communication-deficient HeLa cells to obtain transient expression of the connexin proteins. Intercellular coupling was subsequently assessed by examining transfer of Lucifer yellow between cells. All three mutations resulted in impaired intercellular coupling. The mechanistic reasons for the functional inadequacies of the mutated proteins were investigated. First, intracellular trafficking and targeting of the expressed connexins were determined by immunohistochemistry. Mutation W77R was inefficiently targeted to the plasma membrane and retained in intracellular stores whereas the other two were targeted to the plasma membrane. Oligomerization assays showed that connexins M34T and W77R failed to assemble efficiently into hexameric gap junction hemichannels, but the W44C mutation did so. A cell-free translation system showed that the mutated proteins were inserted into microsomal membranes but the mutations have different effects on the post-translational properties of the expressed proteins. The results point to the conclusion that mutations in the transmembrane domains of connexin proteins influence gap junction assembly.
INTRODUCTION
Gap junctions are regions of contact between two cells where channels that allow direct communication between the adjacent cells are formed. Connexins are the protein subunits of gap junctions; hexameric connexin oligomers are arranged in the plasma membrane as connexon hemichannels that dock with partners in neighbouring cells to generate a direct intercellular communication pathway (1). Fourteen connexin isoforms have been identified in mammals, all showing a common topography with cytoplasmic amino and carboxyl terminal regions, a single intracellular loop and two extra-cellular loops (Fig. 1) (2). Recently, an increasing number of mutations in the gene (GJB2) that codes for one of the connexin proteins, Cx26, have been associated with non-syndromal autosomal hearing impairment (NSAHI) (3-9).
Figure 1. Topological distribution of the Cx26 mutations studied. The mutations studied are indicated. Black line, non-conserved region between different connexins; grey line, conserved regions between different connexins; M1-4, transmembrane domains 1-4; EXL1 and -2, extracellular loops 1 and 2; IL, intracellular loop.
Gap junctions are numerous in the cochlea (auditory) and vestibular (balance) portions of the inner ear, where Cx26 is one of the major connexins expressed (10,11). It has been suggested that these gap junctions are involved in the local circulation of potassium between the fluids of the inner ear (12-14). The endolymph fluid that bathes the apical surface of the sensory hair cells, has a high potassium concentration which is essential for signal transduction by hair cells (11,15,16). However, the high potassium level is present both in the cochlea and vestibular system but patients with Cx26 mutations show only hearing impairment; there does not seem to be a vestibular phenotype. Cochlear endolymph, but not vestibular endolymph, also exhibits a high positive electrical potential, the endocochlear potential (EP). This is thought to be important for providing the energy to power mechanical processes that occur uniquely in the mammalian organ of Corti (auditory epithelium) in response to sound, and underlie the exquisite sensitivity and fine frequency discrimination that is a property of mammalian hearing. EP is generated separately from the high potassium level in endolymph, and during development its initial onset and rise correlates temporally with the formation and increase in size of gap junction plaques in the particular ion-transporting epithelium of the cochlea, the stria vascularis (15). It has been suggested that EP is maintained by local circulation of potassium (12-17) and a role for gap junctions in providing a cell-cell pathway for entry of potassium to the stria vascularis is a strong possibility. If this were so, then mutations in genes for gap junction channel proteins would be expected to have a significant effect on hearing sensitivity but not on the vestibular system.
The most common mutation of the Cx26 gene that is associated with deafness, 35delG (30delG), introduces a stop codon at amino acid position 13 in the N-terminus of the protein (6,18). Another common mutation, 167delT, introduces a premature stop codon in the first extracellular loop at amino acid 56 (8,9,19). These short stretches of amino acids are unlikely to result in oligomeric products arranged as membrane channels. Other mutations, however, are missense mutations distributed throughout the Cx26 protein (6,8,18,20). That such specific mutations cause recognizable functional defects indicates that they occur at functionally important sites. Analysis of the properties of these defective connexin proteins therefore provides a means to identify the significance of those sites in the assembly and functioning of gap junction intercellular channels.
The properties of some mutant Cx26 proteins have been examined in the Xenopus oocyte expression system (21,22), where intercellular communication was found to be electrically deficient. This is of interest because one of the mutations studied, M34T, although originally identified as being associated with a dominantly inherited hearing impairment (3), was subsequently found to occur in individuals with normal hearing and was suggested to be a polymorphism (18,23). The results from the Xenopus expression system indicated that the M34T mutation resulted in a functionally defective protein (21). However, trafficking and channel assembly are best analysed in mammalian cells, for differences are observed between mammalian and amphibian systems (24,25). In the present work, we have used non-communicating HeLa cells, that do not transfer the fluorescent dye Lucifer yellow to neighbours across gap junctions, to investigate the ability of three mutations, namely Cx26M34T, W77R and W44C, to form functional gap junction intercellular channels. These mutations produce defects in the first and second transmembrane domains and first extracellular loop of the Cx26 protein, respectively (Fig. 1). Since all three mutations caused deficiencies in intercellular communication, we extended our investigations to confirm the biochemical basis of the altered phenotypes. This involved studies of the insertion of the mutated connexin into membranes, determination of their intracellular location and finally establishment of whether they were able to oligomerize into hexameric gap junction hemichannels (connexons). The results show that the M34T mutation results in a protein that is trafficked to the plasma membrane but does not oligomerize efficiently into connexon hemichannels, whereas the protein derived from the W77R mutation is neither trafficked to the plasma membrane nor able to oligomerize efficiently.
RESULTS
The positions of the amino acid substitutions caused by the three different mutations in the Cx26 gene studied are illustrated in Figure 1. M34T is positioned in the first transmembrane domain, W44C in the first extracellular loop and W77R in the second transmembrane domain of Cx26. As these amino acids are highly conserved among the different connexins, as determined by ClustalV sequence alignments, it is likely that such single point mutations may impinge on the biogenesis of gap junction intercellular channels constructed of these mutated connexins.
Functionality of cells expressing mutated connexins
To examine whether the mutant connexins could form functional gap junctions, the intercellular transfer of Lucifer yellow was analysed in HeLa cells transiently expressing the mutated connexins. Intranuclear injection of the relevant connexin cDNA into colonies of 10-20 HeLa cells results in connexin expression and gap junction formation within 24 h, as assessed by dye transfer (26). Non-transfected HeLa cells, used as a negative control, displayed negligible basal intercellular coupling (2.2%). With cells expressing wild-type Cx26 (wtCx26), 20% of cells were coupled. The cells expressing the various connexin mutations showed a range of functional properties (Table 1). No dye transfer was observed in cells expressing the W44C mutation. Mutations M34T and W77R showed very low dye transfer (5.7 and 4.1%, respectively) compared with wtCx26. Indeed Student's t-test analysis showed this level of transfer not to be significantly different from non-transfected HeLa cells. The results therefore show that with all three of the Cx26 mutations intercellular communication in HeLa cells was defective.
Table 1. Functionality of Cx26 mutations
| Connexin | Percentage of cells transferring Lucifer yellow |
| WtCx26 | 20.4 ± 5.1 (85) |
| Cx26M34T | 5.7 ± 2.8 (120) |
| Cx26W77R | 4.1 ± 2.4 (90) |
| Cx26W44C | 0 (61) |
| Mock | 2.2 ± 2.2 (54) |
Expression of the mutated connexins in COS-7 cells
Connexin protein expression was analysed in COS-7 cells transfected with the respective cDNAs. Western blot analysis and immunoprecipitation of extracts of cells expressing the mutated connexins showed that proteins of the predicted molecular mass were detected in each instance (Fig. 2). To study targeting of the mutated Cx26 proteins to gap junctions, transfected COS-7 cells were stained with a site-specific antibody directed against the intracellular loop of Cx26 (27). Confocal immunocytochemical analysis showed that cells transfected with wtCx26 gave characteristic punctate plasma membrane staining. Limited staining was also detected intra-cellularly (Fig. 3A). Similar punctate staining at the plasma membrane was also observed with cells expressing the M34T and the W44C connexin mutants indicating that targeting to gap junctions had occurred (Fig. 3B and C). In contrast, the localization of W77R connexin mutation was different with lower levels of plasma membrane staining and evidence for the retention of the protein in intracellular stores in the endoplasmic reticulum (ER) and Golgi apparatus (Fig. 3D). Quantification of immunostaining at the cell periphery indicated that the relative targeting efficiency of the mutated connexins differed. Wild-type Cx26 was efficiently targeted to the gap junction with 53.3 ± 6.7% of the total immunofluorescence detected at the cell periphery. For mutants M34T and W44C the targeting efficiency was lower with 26.9 ± 8.7% and 23.6 ± 4.5% of total immunofluorescence at the cell periphery. With mutant W77R, the targeting efficiency was reduced to 9.8 ± 4% with the remainder of the protein residing inside the cells, especially at areas contiguous to the nucleus. A similar pattern of staining was observed when these mutants were studied in HEK293 cells (data not shown).
Figure 2. Immunoprecipitation of Cx26 mutations. COS-7 cells were transfected with the relevant cDNA and, 48 h post-transfection, the cells were labelled with 35S-promix for 3 h prior to harvesting the cells and immunoprecipitation with an anti-connexin26 antibody. Immunoprecipitates were analysed by SDS-PAGE. Lane 1, mock transfected cells lane 2, wtCx26; lane 3, W44C; lane 4, M34T; lane 5, W77R. The position of Cx26 is indicated.
Figure 3. Immunolocalization of Cx26 mutations. COS-7 cells were transfected with the relevant cDNA and were fixed and stained with a Cx26 antibody 48 h post-transfection. (A) wtCx26; (B) M34T; (C) W44C; (D) W77R. Arrows indicate plasma membrane staining.
Cell-free synthesis of Cx26 mutations
To analyse further the underlying causes of the failure of the mutated connexins to generate functional gap junctions able to exchange Lucifer yellow, the insertion of the proteins into membranes was studied using a cell-free translation system. All three mutated connexin cDNAs were expressed in the coupled in vitro transcription/translation (TNT) system and produced recombinant connexin proteins of the predicted molecular mass (Fig. 4). Aberrant processing in vitro of wild-type Cx26 by signal peptidases occurs concomitantly upon integration into canine pancreatic microsomes and results in the synthesis of connexins lacking a 40 amino acid N-terminal segment extending across the first transmembrane domain (28). The cleaved product has a faster mobility relative to the native Cx26 when analysed by SDS-PAGE (28-30). This proteolytic cleavage was exploited to ascertain whether the mutated connexins were inserted into membranes by supplementing the cell free translation system with canine pancreatic microsomes. Translation in vitro of wild-type Cx26 in the absence of microsomes produces a single 26 kDa product, whereas in the presence of microsomes two bands appear. All three mutated connexins were inserted into microsomal membranes as intact and proteolytic cleaved products (Fig. 4). The results indicate, therefore, that any defects in functionality and/or targeting limitations of the M34T, W44C and W77R mutants are unlikely to arise due to their inability to insert into the membrane.
Figure 4. Insertion of Cx26 mutations into ER membranes. The relevant cDNA was translated in the TNT system in the presence of canine pancreatic microsomes. The reaction products were analysed by 12.5% SDS-PAGE. Lane 1, wtCx26; lane 2, M34T; lane 3, W44C; lane 4, W77R. The positions of full-length Cx26 (Cx26) and the N-terminally cleaved Cx26 (Cx26[prime]) are indicated.
Oligomeric status of connexin26 mutations
The oligomeric status of the Cx26 mutations in transfected COS-7 cells was determined by a velocity sedimentation centrifugation procedure that separates monomeric and oligomeric connexins (27,31,32). The results show that wild-type (wt)Cx26 is efficiently oligomerized into hexameric connexon subunits as >80% of the protein sedimented in the fractions containing hexameric connexons (Fig. 5) (27,30). Similar behaviour was shown by mutation W44C (Fig. 5B). With mutations M34T and W77R, the oligomeric profile in the gradients was altered, with only 15.5 and 8.4%, respectively, of the total connexin protein as hexameric connexons, but 75 and 79%, respectively, of the protein sedimenting in fractions in which intermediate incompletely oligomerized products are found (Fig. 5). In control experiments in which wtCx26 was extracted with SDS, to dissociate connexons into connexins, the connexins were mainly in monomeric form located at the top of the gradients (Fig. 5B). Thus, the results show that wtCx26 and W44C efficiently oligomerized in this system since a proportion sedimented to the area of the gradients where hexamers but the proportion of M34T and W77R forming hexameric assemblies was reduced. The results, summarized in Table 2, show that site-specific mutations in the transmembrane domain of Cx26 impede the ability of the protein to efficiently assemble into hexameric units, with an accumulation of incompletely oligomerized products.
A
 |
B
|
Figure 5. (A) Oligomerization of mutated and wtCx26 in COS-7 cells. COS-7 cells were transfected with the relevant cDNA and connexons were extracted from the cell pellets with a buffer containing dodecyl maltoside (see Materials and Methods) 48 h post-transfection. After separation on 10-40% (w/v) linear sucrose gradients the protein products in each fraction were concentrated and prepared for SDS-PAGE and western blot analysis as described in Materials and Methods. A, wtCx26; B, M34T; C, W77R. `+' represents the sample applied to the gradient. The positions of standard proteins that correspond to connexins or connexons are indicated (29 and 209 kDa, respectively) (27). (B) Relative oligomeric status of Cx26 mutations. The relative amount of Cx26 in each fraction collected from the sucrose gradient was calculated by densitometric analysis of the western blots. Filled bar, hexamer; hatched bar, intermediate oligomeric products; shaded bar, monomer.
Table 2. Properties of Cx26 mutations studied
| Mutant | Domain | Targeting | Functionality | Oligomers | Phenotype | Reference | |
| Dye transfer (mammalain cells) | Electrical (oocytes) | ||||||
| WtCx26 | Yes | Yes | Yes | Yes | Normal | ||
| M34T | TM1 | Yes | No | Noa | Reduced | Debated | 3,18,23 |
| W44C | EL1 | Yes | No | ND | Yes | Dominant | 33 |
| W77R | TM2 | Reduced | No | Nob | Reduced | Recessive | 7 |
aDominant inhibition of wtCx26 coupling (21).
bNo inhibition of wtCx26 coupling (21).
DISCUSSION
To date, 30 different mutations in the GJB2 gene have been reported to be associated with NSAHI. It is estimated that Cx26 mutations may be responsible for up to 50% of cases of NSAHI (7,33) and 10-40% of sporadic cases of congenital deafness (4,6). The biogenesis of gap junction intercellular communication channels involves a series of events resulting in the formation of a hexameric connexon hemichannel embedded in the plasma membrane that docks with a connexon in the membrane of a neighbouring cell (34). The mutations in the Cx26 gene may affect any one or more of these processes.
One role for gap junctions in the inner ear is thought to be in a pathway by which K+ is locally circulated from endolymph to perilymph and back to endolymph via the stria vascularis to help to maintain the high extracellular electrical potential in the cochlea (11,12,15-17). Defects in connexins may therefore reduce the efficiency of K+ circulation and consequently lead to impaired hearing sensitivity. However, although Cx26 is widely expressed in the inner ear, it is now apparent that connexin isoforms in addition to Cx26 are present in the mature mammalian cochlea. Cx30 is widely expressed, and it is associated with the basal cells of the stria vascularis (J. Edwards, D. Becker and A. Forge, manuscript in preparation), a key region in models of how EP is maintained (14,35). Cx31 is also present in the cochlea (J. Edwards, D. Becker and A. Forge, manuscript in preparation) and mutations of the Cx31 gene have also been implicated in NSAHI (36,37). Thus, it is not possible at present to relate mutations in the Cx26 gene to defects in specific physiological processes in the inner ear.
Nevertheless, knowledge of the molecular basis of the Cx26 defects is of importance to an eventual understanding of how these might be overcome. In addition, the fact that mutations at defined sites in the gene result in identified physiological deficits indicates that those sites are of functional importance in the protein molecule. This provides an opportunity to explore structure-function relationships in Cx26. Studies of mutations in Cx32, associated with the peripheral neuropathy Charcot-Marie-Tooth X-linked disease (CMT-X), have allowed Cx mutations to be divided into three categories: group 1 mutations that minimally affect the normal functioning of the protein; group 2 mutations that demonstrate altered communication, especially electrophysiological properties of gap junctions; and group 3 mutations that are functionally deficient owing to major trafficking or targeting abnormalities of the connexins (38).
In the present work the assembly and functionality of three missense mutations in Cx26 associated with NSAHI were investigated using biochemical and cell biological approaches. The results (Table 2) show that the mutated protein subunits examined formed unproductive associations and were unable to form functional homomeric gap junction channels in mammalian cells as efficiently as wtCx26, as determined by their ability to transfer Lucifer yellow to neighbouring cells. Two of the mutations studied, M34T and W44C, located in the first transmembrane and extracellular connexin domains, respectively, were targeted to the gap junction in COS-7 and HEK293 cells. Thus, they fall into the group 2 category described above, i.e. they were assembled and delivered to the plasma membrane but failed to form functional gap junctions. In contrast, a point mutation located in the second transmembrane domain of Cx26 (W77R) was less efficiently targeted and was retained largely in intracellular stores. This behaviour suggests that the protein was misfolded or that the amino acid sequence information that targets the protein to the gap junction was modified. The W77R mutation best fits into the group 3 category of connexin mutations.
To investigate the molecular basis of the biogenetic misfunction, key steps involved in the assembly of gap junctions were investigated. First, the results show that all the mutated connexins were inserted into microsomal membranes. Second, it was evident that mutant W44C oligomerized efficiently into oligomers migrating into the sucrose-detergent density gradient at positions where hexameric channels permeable to small molecules such as ascorbic acid were demonstrated (28,30). In contrast, mutations M34T and W77R resulted in the accumulation of intermediate oligomeric products, suggesting that there were difficulties in assembling into the hexameric connexon channels that form the basis for gap junction formation.
The M34T mutation was originally reported to be associated with a dominantly inherited deafness (3) and has also been associated with a recessive inheritance (8), but it has also been reported in some individuals with normal hearing and was therefore suggested to be a polymorphism (18,23). Our results confirm, from results obtained in two different assay systems, that the protein derived from the Cx26 M34T mutant cDNA is defective. When this mutation is expressed in mammalian cells, the ability to establish gap junction mediated intercellular communication between two cells, assessed by dye transfer methods, is markedly reduced in comparison with cells that express the wild-type protein. One underlying cause of this deficiency appears to be a defect in the ability of the mutant protein to oligomerize into hexameric connexon hemichannels, although the defective protein was targeted to the plasma membrane. Previous work in paired Xenopus oocytes also showed that this mutant was unable to form functional junctions and that it inhibited wtCx26 coupling in a dominant manner, thus explaining the dominant nature of the mutation (21). A similar mutation, M34T, present in Cx32 associated with CMTX patients, was extensively characterized by electro-physiological approaches and was shown to have altered channel gating properties (39). The functional defect was concluded to be due to a large reduction in open channel probability but the mutation did not cause substantial changes in the conductance of the fully open channel (39).
Mutation M34T is present in the first transmembrane domain, a region involved in voltage sensor gating in connexins (40,41) and one that may contribute partially to the wall of the transmembrane channel (39). One explanation for this behaviour is that the mutation alters protein conformation, thus impeding the radial association of connexin subunits around a central channel. Previous work using connexins fused to the Ca2+ reporter protein aequorin showed that their partial oligomerization, initiated in the ER, was sufficient to allow onward trafficking to the plasma membrane and that oligomerization is a major requirement for movement of the connexons from intracellular stores to the plasma membrane and gap junctions (32). The present data suggest that despite the low levels of oligomerization of M34T, sufficient hemichannels are generated to permit limited onward trafficking to the plasma membrane. It is also evident that this Cx26 mutant can co-oligomerize with wtCx26 subunits resulting in dominant inhibition of wtCx26, as observed in paired Xenopus oocytes (21).
Mutation W77R, associated with a recessive form of NSAHI (7) is located in the second transmembrane domain of Cx26. This tryptophan is highly conserved in all connexins suggesting that it may have a crucial stabilizing property on these channel-forming proteins. The present work shows that this mutant had reduced functionality in mammalian cells and it was not efficiently targeted to the gap junction. Furthermore, the mutated connexin showed limited oligomerization into connexon hemichannels. Two independent studies have shown that this mutation is also non-functional in the Xenopus system although it does not affect coupling of wtCx26 channels (21,22). The present studies show, in addition, that this mutation has several effects on gap junction biogenesis. The efficiency of delivery to the gap junction of the mutated connexin is altered, suggesting that a targeting motif is modified or that the protein is misfolded. However, the recessive nature of the mutation and the results from Xenopus oocytes suggest that these inadequacies can be overcome by co-oligomerization with wtCx26 (21), emphasizing the importance of knowledge of the oligomerization competency. That this mutation and M34T, which both affect transmembrane domains, result in defective oligomerization suggests that these regions of the protein are important for oligomerization of the connexins into connexons with functional potential.
The mutation W44C is associated with dominantly inherited NSAHI (33). Tryptophan 44 is a highly conserved amino acid in all connexins. The two extracellular loops possess highly conserved cysteine residues that allow intramolecular disulphide bond formation (42) and are crucial for maintaining the structural integrity of the gap (43). The functional deficiency of W44C mutations can be explained by the introduction of a third cysteine which would impede precise disulphide bond formation and hence may modify the three-dimensional structure of the protein (44).
Although it is generally assumed that all connexins traffic from the ER to the plasma membrane via the classical secretory pathway, evidence is accumulating that Cx26 can be delivered to gap junctions via an alternative pathway that bypasses the Golgi apparatus (27,32). This brefeldin A-insensitive targeting pathway between connexin intracellular stores and the plasma membrane that is followed by Cx26 was shown to be 2- to 3-fold faster than that of Cx32 and Cx43 trafficking (45). An alternative pathway may be important for maintenance of Cx26 gap junctions in the inner ear and in facilitating K+ circulation. It remains to be seen whether any of the Cx26 mutations associated with NSAHI influence the routing and kinetic characteristics of the trafficking of the mutated connexins.
The present work reinforces the general view that specific single point mutations can produce profound effects on channel functioning and assembly. With the Cx26 mutations now studied, intracellular assembly and targeting to gap junctions are disrupted and the efficiency of intercellular communication is compromised. In CMT-X, a series of point mutations severely modify the trafficking and assembly of Cx32, with build up of incomplete oligomers in the secretory pathway (46-48). Trafficking difficulties, with possible channel protein assembly implications similar to those described in the present work, have been reported in the plasma membrane chloride channel in cystic fibrosis (49,50) and with voltage gated channels (51).
In conclusion, the present studies show, using a mammalian model system that three well characterized mutations in Cx26 associated with dominant or recessive NSAHI result in loss of gap junction mediated intercellular communication. The defects observed in the maturation of these mutated connexins into functional gap junction channels may help to clarify further the role that Cx26 plays in the pathophysiology of hearing impairment.
MATERIALS AND METHODS
Construction of Cx26 mutations
Three mutations were introduced into the open reading frame of Cx26 (52) in the vector PCR3 (Invitrogen, Groningen, The Netherlands) containing cytomegalovirus immediate early (CMV) and T7 promoters. For single base pair changes, the Stratagene Quickchange site directed mutagenesis kit (Stratagene, La Jolla, CA) was used according to manufacturer's instructions. The primers used were as follows and the mutated codon is underlined:
M34T forward primer: 5[prime] C TTC ATC TTC CGC ATC ACG ATC CTC GTG GTG GCC G;
M34T reverse primer: 5[prime] C GGC CAC CAC GAG GAT CGT GAT GCG GAA GAT GAA G;
W44C forward primer: 5[prime] GCC GCG AAG GAG GTG TGC GGA GAT GAT GAG CAA GCC;
W44C reverse primer: 5[prime]GGC TTG CTC ATC TCC GCA CAC CTC CTT CGC GGC;
W77R forward primer: 5[prime] C TCT CAC ATC CGG CTC CGG GCT CTG CAG CTG ATC;
W77R reverse primer: 5[prime] G ATC AGC TGC AGA GCC CGG AGC CGG ATG TGA GAG.
PCR products were directly transformed into competent Escherichia coli (XL-Blue) and several colonies selected and sequenced using the PRISM Dye Terminator Cycle Sequencing kit (Perkin Elmer, Warrington, UK) to confirm that the correct base pair changes had been made and that no further mutations were present.
In vivo expression and cellular localization. COS-7 cells (ECACC, Wiltshire, UK) were transfected with vectors encoding the above Cx26 mutations and harvested for western blot analysis as described (24). Proteins were characterized using a primary rabbit antibody generated against amino acid sequences in the intracellular loop of human Cx26 (Gap 28H amino acids 113-124) (27) and secondary goat anti-rabbit antibody conjugated to horse-radish peroxidase (HRP; Bio-Rad, Hercules, CA). Blots were developed using the enhanced chemiluminescence system (Pierce, Rockford, IL).
Immunoprecipitation. COS-7 cells were transfected as described (24). Forty-eight hours post-transfection cells were incubated for 1 h in methionine-free medium (Gibco BRL Life Technologies, Paisley, UK). The cells were pulsed by incubation with 50 µCi 35S-Promix (Amersham Pharmacia Biotech, St Albans, UK) for 3 h at 37°C, washed twice in phosphate-buffered saline (PBS) and harvested in ice-cold immunoprecipitation buffer containing protease inhibitors (53). Wild-type and mutated Cx26 proteins were immunoprecipitated using Cx26 antibodies described above. Connexin-antibody complexes were then solubilized in 3% solubilizing buffer prior to SDS-PAGE analysis followed by fluorography and autoradiography.
Immunolocalization. COS-7 cells (5 × 105) were transfected with 0.5 µg of the relevant Cx cDNA and were fixed 48 h post-transfection in 4% formaldehyde and permeabilized with 0.1% Triton X-100 in PBS as described (24). The subcellular localization of the chimeric proteins was determined using antibody Gap28H (see above). Goat anti-rabbit antibody conjugated to FITC (Amersham) was used as the secondary antibody. Cells were mounted under fluorsave (Calbiochem, Nottingham, UK) and viewed on a Leitz DMBRE confocal microscope. The relative efficiency of targeting of the various connexins to the gap junction was determined by quantification of immunostaining using Adobe Photoshop software.
Functional analysis of cells expressing mutated Cx26
Functionality of the mutated connexins was determined by microinjecting relevant cDNAs into the nucleus of every cell in a colony of contiguous HeLa cells (ECACC) followed, 24 h later, by microinjection of Lucifer yellow (5% in 0.3 M LiCl) into the cytoplasm of a single cell in a colony (26). Transfer of dye between cells expressing the relevant cDNA was determined using filter sets 05 (395-440/460-470 nm; Zeiss, Jena, Germany) on a Zeiss Axiostat microscope. The functionality of each mutant was tested in tested in triplicate with transfer of dye from at least 20 cells per experiment examined. Mock cells were injected with PBS and the efficiency of intranuclear microinjection was analysed by microinjection of cDNA to enhanced green fluorescent protein (Clontech, Palo Alto, CA) which allowed rapid visualization of cells expressing the protein using filter set 09 (450-490/510-520 nm) (26).
Analysis of Cx26 synthesis in a cell-free system. The ability of the mutated connexins to insert into membranes was assessed using a coupled in vitro TNT system supplemented with canine pancreatic microsomes and used as described previously (30). Proteins were solubilized in 2× solubilizing buffer and analysed on 12.5% SDS-polyacrylamide gels (54), fixed, enhanced and vacuum dried prior to autoradiography.
Analysis of the oligomeric state of mutated connexons
COS-7 cells (1 × 107) were seeded onto 10 cm dishes and transfected with 8 µg of the relevant connexin cDNA (24). Ten plates (1 × 108 cells) were used for each assay. Forty-eight hours post-transfection, cells were washed in PBS, harvested in 1 ml PBS and the cells pelleted by centrifugation at 1000 g for 5 min. Connexons were extracted from the cell pellet with 500 µl of a buffer containing 20 mM triethanolamine (TEA) pH 9.2, 20 mM EDTA, 2% w/v dodecyl maltoside and 10 mM DTT (this buffer does not dissociate connexons), in an orbital rotator at 48°C for 1 h (55). As a control, extraction was performed using 20 mM TEA pH 9.2, 20 mM EDTA, 2% w/v SDS and 20 mM DTT to dissociate the connexons into connexins. The reaction mix was centrifuged at 12 000 g for 15 min at 4°C, the supernatants analysed by centrifugation on 10-40% w/v linear sucrose gradients (27). The position of monomeric and oligomeric connexins in the gradients was determined by SDS-PAGE of each fraction followed by western blot analysis and densitometric analysis using the BIORAD 700 densitometer and software (27). The positions of hexameric (connexons) (>28% w/v sucrose), intermediate oligomers [18-28% (w/v) sucrose] and monomeric (connexins; <18% w/v sucrose) products (45) was determined from gradient calibration using standard molecular weight markers.
ACKNOWLEDGEMENTS
We thank Mrs G. Blundell for Research assistance, and C. George for helpful discussion. This work was supported by an MRC programme grant to W.H.E. and a Wellcome Trust project grant to A.F.
REFERENCES
+To whom correspondence should be addressed. Tel: +44 1222 742802; Fax: +44 1222 744905; Email: wmbpem{at}cardiff.ac.uk
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 H. Hibino and Y. Kurachi Molecular and physiological bases of the k+ circulation in the Mammalian inner ear. Physiology, October 1, 2006; 21: 336 - 345. [Abstract] [Full Text] [PDF]
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|
 M. Bicego, M. Beltramello, S. Melchionda, M. Carella, V. Piazza, L. Zelante, F. F. Bukauskas, E. Arslan, E. Cama, S. Pantano, et al. Pathogenetic role of the deafness-related M34T mutation of Cx26 Hum. Mol. Genet., September 1, 2006; 15(17): 2569 - 2587. [Abstract] [Full Text] [PDF]
|
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 Y. Zhang, W. Tang, S. Ahmad, J. A. Sipp, P. Chen, and X. Lin Gap junction-mediated intercellular biochemical coupling in cochlear supporting cells is required for normal cochlear functions PNAS, October 18, 2005; 102(42): 15201 - 15206. [Abstract] [Full Text] [PDF]
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K Cryns, E Orzan, A Murgia, P L M Huygen, F Moreno, I del Castillo, G Parker Chamberlin, H Azaiez, S Prasad, R A Cucci, et al. A genotype-phenotype correlation for GJB2 (connexin 26) deafness J. Med. Genet., March 1, 2004; 41(3): 147 - 154. [Abstract] [Full Text] [PDF]
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N. K. Marziano, S. O. Casalotti, A. E. Portelli, D. L. Becker, and A. Forge Mutations in the gene for connexin 26 (GJB2) that cause hearing loss have a dominant negative effect on connexin 30 Hum. Mol. Genet., April 15, 2003; 12(8): 805 - 812. [Abstract] [Full Text] [PDF]
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 A. Oshima, T. Doi, K. Mitsuoka, S. Maeda, and Y. Fujiyoshi Roles of Met-34, Cys-64, and Arg-75 in the Assembly of Human Connexin 26. IMPLICATION FOR KEY AMINO ACID RESIDUES FOR CHANNEL FORMATION AND FUNCTION J. Biol. Chem., January 10, 2003; 278(3): 1807 - 1816. [Abstract] [Full Text] [PDF]
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 M. Bitner-Glindzicz Hereditary deafness and phenotyping in humans Br. Med. Bull., October 1, 2002; 63(1): 73 - 94. [Abstract] [Full Text] [PDF]
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I. Gottfried, M. Landau, F. Glaser, W.-L. Di, J. Ophir, B. Mevorah, N. Ben-Tal, D. P. Kelsell, and K. B. Avraham A mutation in GJB3 is associated with recessive erythrokeratodermia variabilis (EKV) and leads to defective trafficking of the connexin 31 protein Hum. Mol. Genet., May 16, 2002; 11(11): 1311 - 1316. [Abstract] [Full Text] [PDF]
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 P. E. M. Martin, G. Blundell, S. Ahmad, R. J. Errington, and W. H. Evans Multiple pathways in the trafficking and assembly of connexin 26, 32 and 43 into gap junction intercellular communication channels J. Cell Sci., January 11, 2001; 114(21): 3845 - 3855. [Abstract] [Full Text] [PDF]
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F. Rouan, T. W. White, N. Brown, A. M. Taylor, T. W. Lucke, D. L. Paul, C. S. Munro, J. Uitto, M. B. Hodgins, and G. Richard trans-dominant inhibition of connexin-43 by mutant connexin-26: implications for dominant connexin disorders affecting epidermal differentiation J. Cell Sci., January 6, 2001; 114(11): 2105 - 2113. [Abstract] [Full Text] [PDF]
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M. J Houseman, L. A Ellis, A. Pagnamenta, W.-L. Di, S. Rickard, A. H Osborn, H.-H. M Dahl, G. R Taylor, M. Bitner-Glindzicz, W. Reardon, et al. Genetic analysis of the connexin-26 M34T variant: identification of genotype M34T/M34T segregating with mild-moderate non-syndromic sensorineural hearing loss J. Med. Genet., January 1, 2001; 38(1): 20 - 25. [Abstract] [Full Text]
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